Abstract: In order to find out the potential influencing mechanism of heavy metal Cu on the biogas production during anaerobic fermentation, experiments were designed to study the effects of Cu content in the pig manure (as mass fraction of dry pig manure) on hydrolytic enzyme activities and gas production at mesophilic condition (37℃) in the anaerobic fermentation process. The results indicated that the average cellulase activities and sucrase activities by CK treatment (the mass fraction of Cu was 49.70 mg/kg) were significantly higher than that by H treatment (the mass fraction of Cu was 949.70 mg/kg) during the whole fermentation process, and the average urease activities of CK treatment and L treatment (the mass fraction of Cu was 349.70 mg/kg) were significantly higher than that of M treatment (the mass fraction of Cu was 649.70 mg/kg) and H treatment (p<0.05). Apparently, the copper of H treatment inhibited the gas production. Daily biogas yield of H treatment was below those of CK, L and M treatments within 34 days early in the process, and total biogas production of CK treatment was 9% higher than that of H treatment. Correlation analyses demonstrated that the activities of cellulase, sucrase and urease of the CK treatment, the urease activities of the L treatment and the sucrase activities of the M treatment were significantly positive related to the biogas production rate (p<0.05)，while these relativity of H treatment were not obvious. The results showed that copper might lead to the comprehensive effects of gas production rate through many factors, such as microbial activity, which made no significant correlation between hydrolytic enzyme activities and gas production rate. The results can provide a reference for improving the gas production efficiency of anaerobic fermentation and utilization efficiency of livestock and poultry manure resources.